N-cyanoamidine derivatives as anti-influenza agents

ABSTRACT

Antiinfluenza A activity was found in compounds of the formula: ##STR1## wherein X is ##STR2## where one of R 1  and R 2  is H and the other is phenylamino and Z is --SCH 3  or a pharmaceutically acceptable salt thereof.

This is a division of application Ser. No. 08/322,626, filed Oct. 13,1994, now U.S. Pat. No. 5,475,012 which is a divisional of applicationSer. No. 08/047,730, filed Apr. 15, 1993, now U.S. Pat. No. 5,380,734.

FIELD OF INVENTION

This invention relates to antiviral activity of several novel cyanoiminocompounds. Belshe and Hay, J. Respiratory Diseases 10:552-561, 1989,hypothesize that the M2 protein of influenza virus and the SH protein ofrespiratory syncytial viruses may function as ion channels. Severalcompound possessing the N-cyanoamidine moiety, analogs of a knownpotassium channel opener, pinacidil(N-cyano-N'-4-pyridinyl-N"-1,2,2-trimethylpropylguanidine), were testedand found to have antiviral activity against influenza A virus but wereinactive against respiratory syncytial virus.

DESCRIPTION OF THE INVENTION

The novel compounds useful in this invention are represented by FormulaI below: ##STR3## wherein: X is 2-phenylethyl, 2-furanylmethyl, or##STR4## where one of R¹ and R² is H and the other is phenylamino;

Z is --SCH₃ when X is ##STR5## or when X is 2-phenylethyl or2-furanylmethyl; and the pharmaceutically acceptable salts thereof.

The term "pharmaceutically acceptable salts" encompasses hydrates,solvates, and acid addition salts formed from a basic compound ofFormula I and a pharmaceutically acceptable inorganic or organic acidincluding but not limited to hydrochloric, hydrobromic, sulfuric,phosphoric, acetic, fumaric, succinic, maleic, tartaric, citric andethanesulfonic acids.

The Formula I compounds where Z is a 2-methyl-2-(2-pyridinyl)phenylethylare prepared by reacting an isothiocyanate of the formula X--N═C═S whereX is 2-furanylmethyl or 2-phenylethyl with the lithio derivative of1-phenyl-2-(2-pyridinyl)propane (α-methyl-β-phenyl-2-pyridineethane)followed by reaction with methyl iodide and then reacting theintermediate iminothioic acid methyl ester thus formed with cyanamide asshown in Scheme I below. ##STR6##

The Formula I compounds where Z is --SCH₃ are prepared by reacting anamine of the formula X--NH₂ where X is ##STR7## withdimethyl(cyanoimido)dithiocarbonate as shown in Scheme II. ##STR8##

The amine of the formula X--NH₂ used in Scheme II is prepared byreacting 2-(or 6)-anilinopyridine-3-methanol with 2-aminoethanethiolhydrochloride in 48% hydrobromic acid.

The following synthetic procedures are included for illustrativepurposes only and are not intended to be limiting in any way to thisdisclosure which is limited only by the scope of the appended claims.

EXAMPLE 1

N'-Cyano-α-methyl-N-(2-phenylethyl)-α-(phenylmethyl)-2-pyridineethanimidamide.

(A) α-Methyl-β-phenyl-2-pyridineethane

A solution of 2-ethylpyridine (6.43 g, 60 mmol) dissolved in 100 mL ofanhydrous tetrahydrofuran under nitrogen was cooled to -35° C., andtreated (via syringe) with 2.20N n-butyllithium/hexane (27.3 mL, 60mmol). The solution was stirred at -20° (±5) C. for 25 minutes, treatedat -40° C. (via syringe) with benzyl bromide (10.26 g, 7.14 mL, 60mmol), warmed to 25° C., and stirred for 30 minutes. The solution wasplunged into a stirred mixture of 300 mL of chloroform and 150 mL of icewater, and the organic layer was separated. The aqueous layer wasextracted with 100 mL of chloroform and the combined organic extractswere washed with 150 mL of brine, dried (Na₂ SO₄), and concentrated invacuo to afford the intermediate used in the following reaction withoutfurther purification.

(B)α-Methyl-N-(2-phenylethyl)-α-(phenylmethyl)-2-pyridineethaniimidothioicacid, methyl ester

A solution of α-methyl-β-phenyl-2-pyridineethane (5.13 g, 26 mmol) in 50mL of anhydrous tetrahydrofuran at -35° C. under nitrogen was treated(via syringe) with 25 mmol (11.14 mL) of 2.20N n-butyllithium/hexane andstirred at -25° (±5) C. for 30 minutes. The solution was transferred bya cannula over a 20 minute period into a solution of 2-phenylethylisothiocyanate (4.41 g, 27 mmol) in 25 mL of anhydrous tetrahydrofuranat -70° C. under nitrogen. The mixture was warmed to 25° C. over 1 hour,stirred 30 minutes, cooled (0° C.), treated (via syringe) with methyliodide (3.97 g, 1.75 mL, 28 mmol), and stirred at 25° C. for anadditional 30 minutes. The cooled (0° C.) solution was poured into astirred mixture of 300 mL of chloroform and 150 mL of ice water, and theorganic layer was separated. The aqueous layer was extracted with 100 mLof chloroform and the combined organic extracts were washed with 150 mLof brine, dried (Na₂ SO₄), and concentrated in vacuo to afford the crudeintermediate which was used without further purification.

(C) Title compound

The above material was dissolved in 30 mL of methylene chloride andtreated with cyanamide (1.26 g, 30 mmol) in 12 mL of ether. Additional700 mg portions of cyanamide in ether were added after 18 hours andafter 26 hours. After a total of 40 hours, the solution was filteredthrough alumina (eluted with 10% acetone/chloroform), concentrated invacuo, and triturated from cold ether. Recrystallization from ethylacetate/hexane afforded 5.07 g (55.0%) of the title compound as a whitesolid; mp 162.5°-163.5° C.

Analysis: Calculated for C₂₂ H₂₀ N₄ : C, 78.23; H, 6.56; N, 15.20 Found:C, 78.01; H, 6.53; N, 15.10

EXAMPLE 2

N'-Cyano-N-(2-furanylmethyl)-α-methyl-α-(phenylmethyl)-2-pyridineethanimidamide.

(A)N-(2-Furanylmethyl)-α-methyl-α-(phenylmethyl)-2-pyridineethanimidothioicacid, methyl ester

A solution of 2-(1-methyl-2-phenylethyl)pyridine (6.12 g, 31 mmol) in 60mL of anhydrous tetrahydrofuran under nitrogen at -35° C. was treated(via syringe) with 2.50N n-butyllithium/hexane (30 mmol), and stirred at-25° (±5) C. for 30 minutes. The mixture was transferred via a cannulaover a 15-minute period into a cooled (-70° C.) solution of2-furanylmethyl isothiocyanate (prepared in 89% yield from2-furanylmethylamine by the procedure of J. C. Joehims and A. Seeliger,Angew. Chem. Int. Ed. Engl. 6, 174-175 (1967) (4.45 g, 32 mmol) in 25 mLof anhydrous tetrahydrofuran under nitrogen. The solution was warmed to25° C. over one hour, maintained at room temperature for 30 minutes,cooled (0° C.), and treated (via syringe) with methyl iodide (4.68 g,2.05 mL, 33 mmol). After 30 minutes at room temperature, the cooled (0°C.) solution was poured into a stirred mixture of 300 mL of chloroformand 150 mL of ice water. The organic layer was separated, and theaqueous solution was extracted with 150 mL of chloroform. The combinedorganic extracts were washed with 150 mL of brine, dried (Na₂ SO₄) andconcentrated in vacuo to afford the crude intermediate used belowwithout further purification.

(B) Title compound

The crude intermediate was dissolved in 30 mL of methylene chloride andtreated with cyanamide (1.68 g, 40 mmol) in 15 mL of ether. After 18hours, an additional 800 mg of cyanamide was added in ether, and after72 hours the solution was filtered through alumina (eluted with 10%acetone/chloroform) and chromatographed on silica gel (chloroform, then5% acetone/chloroform as eluents). The product was triturated from coldether and recrystallized from 2-propanol to afford 2.51 g (14%) of thetitle compound as a white solid; mp 152.5°-153.5° C.

Analysis: Calculated for C₂₁ H₂₀ N4O: C, 73.24; H, 5.85; N, 16.27 Found:C, 73.56; H, 5.91; N, 16.27

EXAMPLE 3

N'-cyano-N-[2-[[[2-(phenylamino)-3-pyridinyl]methyl]thio]ethyl]-carbamimidothioicacid methyl ester.

(A) 2-(Phenylamino)-3-pyridinecarboxylic acid

To a suspension of 31.5 g (0.2 mole) of 2-chloro-3-pyridinecarboxylicacid in 75 mL of xylene was added 18.6 g (0.2 mole) of aniline, and thereaction mixture was heated at 190° C. (oil bath) for 45 min; thencooled. The xylene was filtered, and the reaction melt that had formedwas crushed and partitioned between 100 mL of aqueous NaHCO₃ (pH 8) andCH₂ Cl₂ (150 mL). The solid that remained suspended in the two-phasesystem was filtered and discarded. The filtrate was concentrated to 200mL, and the solid that precipitated from the two-phase system wasfiltered to give 7.6 g (13%) ofN-phenyl-2-(phenylamino)-3-pyridinecarboxamide. Acetic acid was added tothe filtrate to acidify the aqueous layer to pH 6 and the CH₂ Cl₂ layerwas separated. The aqueous solution was extracted with CH₂ Cl₂ and thecombined extracts were concentrated to 100 mL; the solid thatprecipitated was filtered and washed sequentially with water and CH₂ Cl₂to give 15.6 g of the product. An additional 5.3 g of the product was toobtained from the filtrate for a total yield of 20.9 g (49%). Thecombined product was triturated with CH₂ Cl₂ and then filtered to give19.6 (46%) of the product as a yellow solid; mp 152°-155° C.; TLC (C₆H₁₂ -EtAc; 4:1 and EtAc-MeOH-NH₄ OH, 16:3:1) showed a single component.MS (EI) m/e (relative intensity): 214 (M+, 100), 213 (94), 195 (70), 169(45), 168 (70), 167 (22), 77 (37), 51 (26).

Analysis: Calculated for Cl₂ H₁₀ N₂ O₂ : C, 67.28; H, 4.71; N, 13.08Found: C, 67.15; H, 4.67; N, 13.07

(B) 2-(Phenylamino)-3-pyridinemethanol

To a refluxing solution of 6.5 g (30.4 mmole) of2-phenylamino-3-pyridinecarboxylic acid and 7.5 mL (64.2 mmoles) oftrimethylborate in 40 mL of THF under N₂ was added dropwise, 65 mL (130mmole) of 2M borane-methyl sulfide over a 30 min period. The reactionmixture was refluxed for 2 hrs, then cooled, and 50 mL of CH₃ OH wasadded dropwise with stirring over a period of 30 min. The mixture wasrefluxed for 5 min, and the solvents were evaporated to leave a gummylight yellow semi-solid which was partitioned between 250 mL of 2N HCland 150 mL of CH₂ Cl₂. The aqueous layer was separated and brought to pH8 (NaHCO₃), and the mixture was extracted with CH₂ Cl₂ (700 mL). The CH₂Cl₂ was evaporated to leave 3.0 g of semi-solid which was purified bydry-column chromatography (glass column, 4×60 cm; 235 gm of silica;eluted with CH₂ Cl₂). The fractions containing the product wereextracted with CH₂ Cl₂ then with CH₃ OH. The solvents were evaporated,and the oily residue was convened to the HCl salt in acetone-Et₂ O togive 1.4 g (40%) of cream-colored solid; mp 180°-182° C. (J. Med. Chem.11, 894 (1968), mp 189°-190° C.). MS (EI) m/e (relative intensity): 200(M+, 27), 199 (24), 182 (65), 181 (100).

Analysis: Calculated for Cl₂ H₁₃ ClN₂ O: C, 60.89; H, 5.54; N, 11.84Found: C, 60.48; H, 5.57; N, 11.77

(C) 2-[[[2-(phenylamino)-3-pyridinyl]methyl]thio]ethylamine

A solution containing 8.0 g (40.0 mole) of2-(phenylamino)-3-pyridinemethanol and 5.0 g (44.0 mmole) of2-aminoethanethiol hydrochloride in 80 mL of 48% HBr was refluxed for 25hrs, then cooled. The solvent was evaporated in vacuo to leave a residuewhich was stirred with 100 mL of H₂ O, and the insoluble solid thatremained was filtered, washed (H₂ O, then with petroleum ether, 30°-60°C.) and air-dried to give 11.3 g (67%) of the dihydrobromide salt; mp187°-292° C. An additional 2.2 g of the product (as the free base) wasobtained from the filtrate upon treatment with K₂ CO₃ (pH 10) andextraction with CH₂ Cl₂ ; total yield (81%).

(D) Title Compound

A solution containing 6.5 g (0.025 mole) of2-[[[2-(phenylamino)-3-pyridinyl]-methyl]thio]ethylamine, 3.7 g (0.025mole) of dimethyl(cyanoimido)dithiocarbonate and 3 ml (0.022 mole) ofEt₃ N in 50 mL of CH₃ CN was stirred at room temperature for 18 hrs,then refluxed for 3 hrs. The progress of the reaction was followed byTLC (CH₂ Cl₂ -MeOH, 10:1). The solvent was evaporated and the dark oilyresidue was purified by chromatography (3.5×70 cm glass column; 240 g ofsilica, eluted with CH₂ Cl₂ -EtAc, (1:1). Fractions containing theproduct were evaporated to give 6.1 g (68%); a 3.5 g sample wastriturated with CH₂ Cl₂, the solid was filtered to give 2.7 g (30%) ofwhite solid; mp 120°-123° C. MS (EI)m/e (relative intensity): 357 (M+,7), 309 (14), 308 (13), 215 (45), 184 (42), 183 (98), 182 (85), 181(100), 77 (23), 48 (44), 47 (56), 45 (32).

Analysis: Calculated for C₁₇ H₁₉ N₅ S₂ : C, 57.12; H, 5.36; N, 19.59Found: C, 56.92; H, 5.37; N, 19.51

EXAMPLE 4

N'-cyano-N-[2-[[[2-(phenylamino)-5-pyridinyl]methyl]thio]ethyl]carbamimidothioicacid methyl ester.

(A) 6-Phenylamino-3-pyridinecarboxylic acid

6-Chloro-3-pyridinecarboxylic acid (15.76 g, 0.1 mole) and aniline (9.3g, 0.1 mole) were heated together in 50 mL xylene at 180°-185° C. (oilbath) for 3 hours. After cooling, the solid that formed was filtered andwashed with Et₂ O. The solid was triturated with NaHCO₃ solution (pH 8),then filtered to give 7.0 g (24%) ofN-phenyl-6-(phenylamino)-3-pyridinecarboxamide. The aqueous NaHCO₃solution was carefully neutralized with glacial HOAc (pH 6), and thesolid that precipitated was filtered and washed (H₂ O and with Et₂ O);yield 12.13 g (57%), mp 264°-266° C. MS (EI) m/e 214 (M+), 213 (M-1,base), 169 (M-45), 167 (M-47).

Analysis: Calculated for C₁₂ H₁₀ N₂ O₂ : C, 67.28; H, 4.71; N, 13.08Found: C, 67.03; H, 4.64; N, 13.01

(B) 6-Phenylamino-3-pyridinemethanol

Following the procedure given in part B of Example 3,6-phenylamino-3-pyridinemethanol was prepared in 78% yield from6-phenylamino-3-pyridinecarboxylic acid; mp 119°-122° C.

(C) 2-[[[6-(phenylamino)-3-pyridinyl]methyl]thio]ethylamine

Following the procedure given in part C of Example 3, the title compoundwas prepared from 6-phenylamino-3-pyridinemethanol and2-aminoethanethiol hydrochloride.

(D) Title compound

A solution containing 4.0 g (15.4 mmole) of2-[[[6-(phenylamino)-3-pyridinyl]methyl]thio]ethylamine, 2.3 g (15.7mmole) of dimethyl(cyanoimido)dithiocarbonate and 1.7 g (16.8 mmole) ofEt₃ N dissolved in 60 ml of MeCN was stirred at room temperature for 15minutes during which time a yellow precipitate formed. TLC (CH₂ Cl₂-MeOH, 10:1) showed that the reaction was complete. The product wasfiltered to give 4.5 g (82%) of a yellow solid, mp 180°-182° C., MS m/e(relative intensity): (CI) 358 (M+1, 8), 183 (22), 128 (100): (EI) 309(23), 183 (100), 182 (52), 77 (34), 48 (36), 47 (58).

Analysis: Calculated for C₁₇ H₁₉ N₅ S₂ : C, 57.12; H, 5.36; N, 19.59Found: C, 57.42; H, 5.40; N, 19.80

The anti-influenza A activities were determined in Madin Darby CanineKidney (MDCK) cells seeded in 96-well tissue culture plates. The testcompounds (0.16-16 μg/ml) were added to the cells 2 hours prior toinfecting the MDCK cells with FluA/WSN (˜25 plaque forming units(PFU)/well). The plates were stained with crystal violet 2 days later toreveal the cytopathic effects. Antiviral activities were determined intriplicate while cellular toxicities were determined in a single test.Tests on the known antiviral agents amantadine and ribavirin were runfor comparative purposes. Activity against respiratory syncytial virus(RSV) was determined similarly in HEp-2 cells except that ˜500 PFU/wellof RSVA₂ were added and the plates were stained 3 days after infection.Cytotoxicity in five different cell lines was determined. The results ofthese assays are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Antiviral and Cytotoxicity Data                                                       Antiviral MI C.sub.50 (μg/ml)                                                           Cytoxicity (minimal toxic dose μg/ml)                 Compound                                                                              A/WSN                                                                              RSVA.sub.2                                                                            MDCK HEp-2                                                                             A549                                                                              MRC-5                                                                             Vero                                    __________________________________________________________________________    Example 1                                                                             0.16-0.5                                                                           --      >50  >50 >50 >50 >50                                     Example 2                                                                             0.16-0.5                                                                           --      >50  >50 >50 >50 >50                                     Example 3                                                                             0.5  --      >100 >100                                                                              >100                                                                              >100                                                                              >100                                    Example 4                                                                             5    --                                                               Amantadine                                                                            16   --      >100 >100                                                                              >100                                                                              >100                                                                              >100                                    Ribavirin                                                                              1.6-5.0                                                                           1.6-5.0 >100 >100                                                                              >100                                                                              >100                                                                              >100                                    __________________________________________________________________________

Strain specificity of the antiinfluenza activities of the compounds ofExamples 1 and 2 against two additional strains of influenza A and onestrain of influenza B were determined. Both compounds were activeagainst A/WSN and A/Kaw but were inactive against A/LA and B/Yama. Theknown antiviral compounds amantadine and ribavirin showed differentspecificities. Results of the strain specificity assays are shown inTable II.

                  TABLE II                                                        ______________________________________                                        Antiinfluenza Strain Specificity                                                     Median Inhibiting Concentration (IC.sub.50 μg/ml)                   Virus.sup.a                                                                            Example 1 Example 2 Amantadine                                                                             Ribavirin                               ______________________________________                                        FluA/WSN 0.16-0.5  0.16-0.5   16      1.6-5                                   FluA/Kaw 0.5-1.6   0.5-1.6   0.5-1.6  >16                                     FluA/IA  >16       >16       0.16     >16                                     FluB/Yama                                                                              >16       >16       >16      1.6-5                                   ______________________________________                                         .sup.a A/WSN was tested in MDCK cells; A/Kaw, A/LLA and B/Yama were teste     in chick kidney primary cultures.                                        

Based on the in vitro antiinfluenza A test data of the inventioncompounds as compared with amantadine, which has a recommended dailydose of 200 mg in a normal human adult under age 65 for the treatment ofinfluenza A, the dosage of a compound of this invention would be in therange of 2 to 200 mg/day which may be given in one dose or divideddoses. The exact dosage will, of course, be determined according tostandard medical principles by a physician.

The compounds may be administered neat or with a pharmaceutical carrierto a mammal in need thereof. The pharmaceutical carrier may be solid orliquid.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredients. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g., glycols) and their derivatives,and oils (e.g., fractionated coconut oil and arachis oil). Forparenteral administration, the carrier can also be an oily ester such asethyl oleate and isopropyl myristate. Sterile liquid carriers are usefulin sterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellant.

For administration by intranasal or intrabronchial inhalation orinsufflation, compounds of this invention can be formulated into anaqueous or partly aqueous solution, which can then be utilized in theform of an aerosol.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministrated intravenously. The compound can also be administeredorally either in liquid or solid composition form.

What is claimed is:
 1. A compound of the formula ##STR9## wherein X is##STR10## where one of R¹ and R² is H and the other is phenylamino; andZ is --SCH₃, or a pharmaceutically acceptable salt thereof.
 2. A methodof treating influenza A infections in mammals which comprisesadministration to the mammal in need thereof of a therapeuticallyeffective amount of a compound of the formula: ##STR11## wherein X is##STR12## where one of R¹ and R² is H and the other is phenylamino; andZ is --SCH₃, or a pharmaceutically acceptable salt thereof.
 3. Apharmaceutical composition for treating influenza A infections inmammals comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of a compound of the formula: ##STR13##wherein X is ##STR14## where one of R¹ and R² is H and the other isphenylamino; and Z is --SCH₃, or a pharmaceutically acceptable saltthereof.
 4. A compound according to claim 1 which isN'-cyano-N-[2-[[[2-phenylamino)-3-pyridinyl]methyl]thio]ethyl]carbamimidothioicacid methyl ester or a pharmaceutically acceptable salt thereof.
 5. Acompound according to claim 1 which isN'-cyano-N-[2-[[[2-phenylamino)-5-pyridinyl]methyl]thio]ethyl]carbamimidothioicacid methyl ester or a pharmaceutically acceptable salt thereof.
 6. Themethod according to claim 2 wherein the compound used isN-[2-[[[2-phenylamino)-3-pyridinyl]methyl]thio]ethyl]carbamimidothioicacid methyl ester or a pharmaceutically acceptable salt thereof.
 7. Themethod according to claim 2 wherein the compound used isN'-cyano-N-[2-[[[2-phenylamino)-5-pyridinyl]methyl]thio]ethyl]carbamimidothioicacid methyl ester or a pharmaceutically acceptable salt thereof.